Excretion of 2- and 3-monomethyl ethers of 2-hydroxyestrogens in healthy male volunteers.

The formation of catecholestrogens by 2- and 4-hydroxylation of monophenolic estrogens represents a major route of estrogen metabolism. In vitro and in vivo studies on catecholestrogens have shown that 2-hydroxylated catecholestrogens are primarily inactivated by O-methylation, while o-methylation of 4-hydroxylated estrogen is of minor importance. In the present study the in vivo production of isomeric 2- and 3-monomethyl ethers of 2-hydroxyestrogens was measured in 12 healthy omnivorous male volunteers aged 51 +/- 4 years. The sum of estrone and 17 beta-estradiol, 2-hydroxyestrogens (sum of 2-hydroxyestrone and 2-hydroxyestradiol), 4-hydroxyestrogens (sum of 4-hydroxyestrone and 4-hydroxyestradiol) and the sum of the isomeric monomethyl ethers of 2-hydroxyestrone and 2-hydroxyestradiol were measured in 24-h urinary samples. The determination included hydrolysis of steroid conjugates, separation by chromatographic steps and final quantification by radioimmunoassay. The specificity of the antibodies enabled differentiation between the isomeric monomethyl ethers. The mean urinary excretion rates were 8.8 +/- 2.9 micrograms/24 h for estrone plus estradiol, 5.2 +/- 2.4 micrograms/24 h for 2-hydroxyestrogens and 1.3 +/- 0.5 micrograms/24 h for the 4-hydroxyestrogens. The 2- and 3-monomethyl ethers of the 2-hydroxyestrogens were found in all individuals, with excretion rates of 5.8 +/- 2.6 micrograms/24 h for 2-methoxyestrogens and 3.6 +/- 1.1 micrograms/24 h for 2-hydroxyestrogen-3-methyl ethers. The findings indicated that 2-hydroxyestradiol is metabolized in vivo by 2-O-methylation and, to a lesser extent, by 3-O-methylation.

Fibronectin is an estrogen-repressed protein in RUCA-I rat endometrial adenocarcinoma cells.

We recently established and characterized two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of charcoal stripped fetal calf serum. We further demonstrated that culturing of these cells on a reconstituted basement membrane induced the estrogen responsiveness for both proliferation and gene expression. Particularly, the expression of components of the complement C3 system, which represent major estradiol inducible proteins in the rat uterus in vivo, were found to be under the control of estrogens and antiestrogens. In this paper the search for estrogen repressed proteins is reported. For this purpose secretory proteins of RUCA-I cells were metabolically labelled with 35S-methionine and tested for the presence of estrogen-repressed, antiestrogen-inducible protein species. Analyzing cell culture supernatants of RUCA-I cells by polyacrylamide gel electrophoresis under reducing conditions a protein with an apparent size of approx. 250-270 kDa became conspicuous. The formation and secretion of this protein was suppressed by estradiol and induced by the antiestrogen ICI 164384. Gel electrophoresis performed under non-reducing conditions and hyaluronidase digestion showed that this estrogen-repressed protein represents a dimeric glycoprotein. By immunoprecipitation this glycoprotein was identified as fibronectin. Investigations of steady state mRNA levels of fibronectin by rtPCR suggested a post-transcriptional regulation of this molecule by estradiol. This is the first report on repression of components of the extracellular matrix by estradiol and induction by the complete antiestrogen ICI 164384. The consequences of this finding in regard to growth and invasion of endometrial tumors are discussed.

Extracellular matrix induces hormone responsiveness and differentiation in RUCA-I rat endometrial adenocarcinoma cells.

We recently described the establishment and the characterization of two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite fairly high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of serum. A limited hormonal response to the antiestrogen tamoxifen was detectable in RUCA-I but not in RUCA-II cells. To advance our cell culture conditions we plated RUCA-I cells on a layer of reconstituted basement membrane (Harbor Matrix) in the presence of a serum-free defined medium. These cell culture conditions induced hormone responsiveness of RUCA-I cells and permitted a stimulation of proliferation by estradiol. Further, two estradiol-induced secretory proteins with an apparent molecular weight of 115 kD and 60 kD could be identified by SDS-gelelectrophoresis if analyzed under reducing conditions. These proteins migrated as a single band in a non-reducing electrophoresis gel and were identified as components of the complement C3 system. Additionally, our results suggest that the effects of extracellular matrix and hormones on the expression of these proteins are additive. We conclude that processes of functional differentiation are most likely to occur in this in vitro model, particularly since the expression of components of the complement C3 system was under estrogenic control. Complement C3 proteins represent major estradiol-inducible secretory protein of the immature rat uterus in vivo. Culturing RUCA-I cells on top of a layer of reconstituted basement membrane provides a novel tool to study the importance of the extracellular environment on the hormone-induced gene expression in endometrial carcinogenesis in vitro.

A novel radioimmunoassay of allopregnanolone.

A radioimmunoassay for determination of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in serum or plasma has been developed and evaluated. The method employs rabbit antiserum to 3 alpha-hydroxy-5 alpha-pregnane-11,20-dione-11-O-carboxymethyloxime bovine serum-albumin conjugate and tritiated radioligand. The main cross-reactant interfering in the assay, progesterone, is eliminated by permanganate oxidation. Two assay variants were compared, with and without a micro-column chromatography. The simplified variant appeared to be reliable enough for determination of allopregnanolone in normally menstruating women at luteal phase, whereas the column-chromatography step is necessary when analyzing samples of expected low analyte concentration as in women in follicular phase, postmenopausal women, or in men. The levels of allopregnanolone in healthy women correlated excellently with progesterone in agreement with previous findings.

Inhibition of human placental sterylsulfatase by synthetic analogs of estrone sulfate.

Synthetic analogs of estrone sulfate which carry differently substituted sulfonyl groups at position 3 of an invariable 3-desoxyestrone (dE1) moiety were tested in vitro as inhibitors of the human placental sterylsulfatase. Using both placental microsomes and a highly purified placental sterylsulfatase preparation as the enzyme source and dehydroepiandrosterone [35S]sulfate or estrone [35S]sulfate as the substrate, the following order of inhibitory potencies was observed: dE1-3-sulfonylchloride > dE1-3-sulfonylfluoride approximately dE1-3-sulfonate > dE1-3-sulfonamide approximately 3-methylsulfonyl-dE1. According to the results, the association of enzyme and inhibitor appears to be favored by an electronegative substituent at the sulfur atom (-C1, -F, -O-). Since, however, even the most potent synthetic inhibitor was bound by the enzyme with significantly lower affinity than was the natural substrate estrone sulfate, an oxygen function between the aromatic ring and the sulfur atom may be necessary for high affinity binding towards the sterylsulfatase. In addition to its fast reversible association with the enzyme, dE1-3-sulfonylchloride further affected the sulfatase activity in a time-dependent manner. This latter inhibitory activity which may be due to a covalent modification (alkylation) of sterylsulfatase by the analog was partially prevented in the presence of substrate.

Androgen ablation induces tenascin expression in the rat prostate.

Tenascin is a glycoprotein of the extracellular matrix of mesenchymal derived tissue compartments. Although the DNA sequences that code for tenascin production are known and the protein structure is well characterized, little is known about regulation of tenascin expression. Therefore, we are interested in hormonal aspects of tenascin expression. In this study, we addressed the question if androgen deprivation and prostatic involution would influence tenascin expression in the prostate. Methodologically, in two series of experiments, intact and orchiectomized testosterone propionate substituted male rats were subjected to one of the following hormonal treatments: a) flutamide, b) the antiandrogen casodex, and c) cyproterone acetate (CPA). As controls in each series, we used untreated controls and orchiectomized rats. After a period of 14 days of treatment, prostates were removed. Tenascin immunostaining of the sectioned specimen from the control and the hormonally treated animals demonstrated the following: 1) Little, if any tenascin immunoreactivity was detectable in prostates of untreated animals. 2) Androgen deprivation with either treatment resulted in tenascin expression in the stroma of the prostates. 3) Tenascin expression appeared to be variable both semiquantitatively and in the staining pattern detectable except in prostates treated with CPA, in which we observed the most uniform and most widespread staining pattern. From these results, we conclude that androgen deprivation induces tenascin expression in the stroma of the involuting prostate. Remodelling of the extracellular matrix of the stroma, known to appear in the process of prostate involution by androgen ablation, represents a process which has not been discussed with tenascin expression so far.

Sterylsulfatase expression in normal and transformed human placental cells.

Isolated cytotrophoblast cells and choriocarcinoma cell lines are commonly applied in-vitro systems for the study of human placental endocrine function. We tested these normal and transformed placental cells for expression of the enzyme sterylsulfatase which is necessary for the production of free steroids from sulfoconjugated precursors in the placenta as well as in other human tissues, and compared the results with respective data obtained from term placental tissue. Specific sterylsulfatase activity was highest in placental homogenates but was lower by about a factor of 5 to 10 in homogenates of freshly isolated cytotrophoblast or JEG-3 cells and by about a factor of 100 in BeWo cell homogenates; the enzyme activity could not be detected in Jar cells. Sterylsulfatase mRNA levels as analyzed by Northern blotting roughly paralleled the levels of enzyme activity measured in cytotrophoblast, JEG-3, and BeWo cells; in Jar cells, RNA species complementary to the specific probe were clearly detectable but differed by size from the mRNA species found in the other cells. Our results indicate that sterylsulfatase activity is differently expressed in normal and transformed placental cells due to different rates or products of gene transcription in these cells.

Stromal expression of tenascin is inversely correlated to epithelial differentiation of hormone dependent tissues.

We were previously investigating the expression of the extracellular matrix glycoprotein tenascin in normal and malignant endometrial tissues of humans and rodents. These studies suggested that the expression of tenascin was induced by proliferating epithelia (normal and particularly malignant) and was downregulated with their differentiation. The aim of this study was to investigate the hormone dependency of tenascin expression in (a) the transplantable EnDA endometrial tumor model with or without estrogen deprivation (ovariectomy) of the animals, (b) DMBA-induced rat mammary tumors with or without a hormonal treatment of the animals [ovariectomy, antiestrogen (tamoxifen) or antiprogestin (ZK 98299) treatment] and (c) in the rat prostate of untreated or androgen deprived animals (orchiectomy, flutamide-, casodex- or cyproterone acetate (CPA)-treatment). 1. Estrogen withdrawal by ovariectomy did not affect tenascin expression in transplantable EnDA endometrial adenocarcinoma, meaning the entire extracellular space of the stromal mesenchyme was decorated by tenascin immunoreactivity. 2. In untreated DMBA-induced rat mammary tumors almost the entire extracellular space of the stroma was stained by tenascin immunoreactivity. Ovariectomy and antiestrogen treatment did not affect tenascin expression. In contrast, antiprogestin treatment induced terminal differentiation of mammary tumor cells and in parallel downregulated tenascin expression. 3. In the normal rat prostate no tenascin was detectable by immunocytochemistry. However, following androgen deprivation we found tenascin expression in the stroma of the prostate. The most prominent expression was observable after CPA-treatment, possibly due to its progestagenic potency. In conclusion, the hormones and antihormones tested show no direct effect on the stromal expression of tenascin. However, proliferative activity and a low degree of differentiation of the epithelium induces tenascin expression, whereas epithelial differentiation apparently shuts down tenascin expression. Preliminary in vitro studies suggest that paracrine acting growth factors trigger the hormonal regulation of tenascin expression.

Catecholestrogens are agonists of estrogen receptor dependent gene expression in MCF-7 cells.

The catecholestrogens, namely 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) are important, naturally occurring metabolites of E2. Here we studied their role on estrogen dependent processes. Using the MCF-7 cell line as a model system we analyzed the potency of 2- and 4-OH-E2 on the synthesis of the 160 kDa secreted protein and on the transcription of the pS2 mRNA. Both processes are known to be E2 inducible and are mediated by the estrogen receptor. Control incubations using E2 and antiestrogens were performed to validate the assay procedure and to enable us to comparatively study the effects of the catecholestrogens. Stimulating MCF-7 cells for 2 days with 10(-8) M 2- or 4-OH-E2 resulted in an induction of the synthesis of the 160 kDa protein and in an increase in pS2 mRNA. Following hormonal stimulation with 2- or 4-OH-E2 [35S]methionine labeling of MCF-7 cells increased the level of newly synthesized and secreted 160 kDa protein 54 and 88% compared with the inductive potency of E2 (100%). The pS2 mRNA in MCF-7 cells was increased by a 2 day treatment with 10(-8) M 2- or 4-OH-E2 by 48 and 79%, respectively, compared to E2. Therefore, we conclude that the estrogen receptor is transcriptionally active in MCF-7 cells upon binding of catecholestrogens. The estrogen receptor in vivo may be active if the intracellular concentration of catecholestrogens generated is sufficient to allow occupation of the receptor. The possible action of these hormones in vivo is discussed.

Weighted serum pools in comparison to the trapezoidal rule for estimating AUCs for ethinyl estradiol. The relationship of the variance of the determination to the interindividual variance.

The concept of a weighted pool for estimating the area under the curve (AUC) is presented and set in relationship to the trapezoidal rule. An example from a pharmacokinetic study on ethinyl estradiol is used to demonstrate the use of variance component analysis for relating the intraindividual variance of the AUC, trapezoidal rule and weighted pool to the variance of the determination process. Depending on the sampling times, the theoretical variance of the weighted pool is greater than the theoretical variance of the trapezoidal rule. In the example presented, it was shown that this difference is of no importance in relation to the interindividual variance of the AUC, which dominates the total variance. In the example study, routine quality control samples were also determined in each assay, which allowed independent confirmation of the discussed results on the intraindividual variance of the AUCs.

Tamoxifen and ZK 119010 exert mixed agonistic and antagonistic effects on pS2 expression in MCF-7 cells.

pS2 is a major estradiol-inducible gene in MCF-7 breast cancer cells. In this study we tested the effects of tamoxifen and ZK 119010, a novel nonsteroidal antiestrogen, on pS2 gene expression with or without a pretreatment of cells with estradiol (10(-11), 10(-8) M). Estradiol increased pS2 expression in MCF-7 cells approximately 12-fold. Tamoxifen (10(-6) M) reduced estradiol-induced pS2 expression to 78% of the stimulated level, while ZK 119010 was 50% effective. Given alone, either of the two antiestrogens in the above concentrations evoked a pS2 gene expression in MCF-7 cells significantly above background levels. From these data we conclude that the antiestrogens tamoxifen and ZK 119010 possess both antagonistic and agonistic potencies in MCF-7 cells. However, the antiestrogenic potency of ZK 119010 seems to be higher than that of tamoxifen.

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to the estrogen receptor of MCF-7 cells in cytosolic extracts and in nuclei of intact cells.

The catechol estrogens (CE), 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) were analyzed for their binding affinity to the estrogen receptor of MCF-7 cells. Applying a competitive binding assay to cytosols prepared from MCF-7 breast cancer cells, we measured a relative binding affinity of 23% (2-OH-E2) and 26% (4-OH-E2) compared to E2. Nuclear binding assays with the same cell line demonstrated a high specific binding with Kd's of 0.31 nM (2-OH-E2) and 0.21 nM (4-OH-E2). The relative binding affinity measured was 25% and 42% for 2-OH-E2 and 4-OH-E2, respectively. Based on this nuclear binding it can be concluded that the estrogen receptor occupied by CE is bound within the nucleus and might therefore be transcriptionally active.

Catecholestrogens are MCF-7 cell estrogen receptor agonists.

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

Pharmacokinetics of gestodene and ethinylestradiol in 14 women during three months of treatment with a new tri-step combination oral contraceptive: serum protein binding of gestodene and influence of treatment on free and total testosterone levels in the serum.

The pharmacokinetics of gestodene (GEST) and ethinylestradiol (EE2) were determined in 14 healthy women (age 18 to 32 years) during a treatment period of three months with a new tri-step combination oral contraceptive (Milvane). Prior to this treatment period, the same women received a single administration of a coated tablet containing 0.1 mg GEST together with 0.03 mg EE2. There was a wash-out phase of one week between both treatments. Following single dose administration, a mean terminal half-life of 18 h was observed for GEST. The total clearance was 0.9 ml x min-1 x kg-1 and the volume of distribution was 84 l. During a treatment cycle, GEST levels in the serum accumulated by a factor of 8 as compared to single dose administration. Steady-state drug levels were reached during the second half of each cycle. As compared to single dose administration, the following changes were observed for GEST at the end of treatment cycles one and three: prolonged terminal half-life (20 to 22 h), reduced total (0.16 ml x min-1 x kg-1) and free clearance (ca. 27 ml x min-1 x kg-1), reduced volume of distribution (ca. 18 l). A concomitant EE2-induced increase in the SHBG concentrations by a factor of three as compared to pretreatment values was observed during a treatment cycle and appeared to be mainly responsible for the changes in the pharmacokinetics of GEST. Marked changes were also seen for the serum protein binding of GEST. After single dose administration, the free fraction of GEST was 1.3% and the fractions bound to SHBG and albumin were 69.4% and 29.3%, respectively. At the end of cycle one, the free fraction was only 0.6% and the fractions bound to SHBG and albumin were 81.4% and 18.0%, respectively. There was no difference in corresponding pharmacokinetic parameters and in the serum protein binding of GEST at the end of cycles one and three. On the last day of treatment cycles one and three, the AUC(0-4h) values of EE2 were 299.2 and 278.1 pg x ml-1 x h, respectively, which corresponds to an about 30% increase as compared to single dose administration, where an AUC(0-4h) value of 216.1 pg x ml-1 x h was found. Total and free testosterone concentrations decreased during treatment cycles one and three by about 36% and 60%, respectively, compared with the corresponding values measured prior to treatment. The fraction of unbound testosterone thus decreased from 0.5% to 0.3% during treatment.

PIP: In Germany, researchers examined blood samples from 14 healthy young women, who first took a single oral dose of 0.1 mg gestodene and .03 mg ethinyl estradiol, then Milvane, a new tri-step combination oral contraceptive, for 3 months, to learn in detail the pharmacokinetics of gestodene. A 1-week wash-out phase occurred between the 2 treatments. The mean terminal half-life for gestodene after receiving the single dose was 18 hours. It volume of distribution stood at 84. Total clearance was 0.9 ml x min-1 x kg-1. Gestodene serum levels accumulated by a factor of 8 when compared to levels after single-dose administration. Drug levels reached a steady-state by the second part of each treatment cycle. At the end of treatment cycles 1 and 3, in comparison with single-dose administration, gestodene had a prolonged terminal half-life (20-22 h), reduced total and free clearance (0.16 ml x min-1 x kg-1 and ca. 27 ml x min-1 x kg-1, respectively), and reduced volume of distribution (ca. 18 1). When comparing single-dose administration and 3-month treatment levels, ethinyl estradiol increased sex hormone binding globulin (SHBG) levels by a factor of 3, apparently causing the changes in the pharmacokinetics of gestodene. Further, the free fraction of gestodene fell from 1.3 to 6%, and the fractions bound to SHBG increased from 69.4 to 81.4% while the fractions bound to albumin fell from 29.3 to 18%. The ethinyl estradiol values of the area under the curve stood at 299.2 pg x ml-1 x h at 0 hours of the last day of treatment cycle 1 and 3. At 4 hours, it was 278.1 pg x ml-1 s h. Between pretreatment and treatment cycles 1 and 3, total and free testosterone levels fell by almost 36% and 60%, respectively, resulting in a decrease of the fraction of unbound testosterone from 0.5 to .03% during treatment. These findings showed that pharmacokinetic parameters did not differ between cycles 1 and 3, indicating that equilibrium had been already reached after one cycle and that prolonged treatment should not result in any other changes.

Localization of tenascin in uterine sarcomas and partially transformed endometrial stromal cells.

Normal mesenchymal cells within developing embryonic organs and transformed stromal cells in organs undergoing spontaneous carcinogenesis have the capacity for normal or altered expression of the extracellular matrix glycoprotein tenascin (Tn). Mesenchymal cell constituents of normal adult organs show only a very limited tendency to deposit Tn in their extracellular matrix. In the present study, we investigated whether malignant human mesenchymal cells derived from uterine sarcomas or normal human endometrial stromal cells partially transformed via transfection with selected oncogenes have the capacity to produce and deposit Tn. We reached the following conclusions: (1) compared with normal endometrial tissues, uterine sarcomas show heterogeneous, but increased, immunoreactive staining patterns exclusively within the extracellular compartment, regardless of the histologic subtype of the tumor; (2) in vitro, all normal and transfected stromal cells and cell lines examined secreted Tn into the tissue culture medium; (3) this secretory ability was not translated into morphologic uniformity, since immunoreactivity detected by confocal laser scanning microscopy was observed in only selected cell populations; (4) also, the deposition and the incorporation of Tn depended upon the density of transfected cells, and (5) double-staining experiments revealed that Tn could always be localized in close proximity to fibronectin. In summary, the production of Tn is increased in most cases of human uterine sarcoma. The capacity of stromal cells to deposit Tn in a matrix-like structure in vitro, rather than increase production of Tn, is correlated with the degree of neoplastic progression.

Functions of estrogens and anti-estrogens in the rat endometrial adenocarcinoma cell lines RUCA-I and RUCA-II.

Inbred rats of the DA/Han and BDII/Han strains have been proposed as suitable model systems for studying hormonal carcinogenesis, because they die mainly from hormone-dependent endometrial adenocarcinoma. Here we characterize the RUCA-I cell line derived from an endometrial adenocarcinoma of an inbred DA/Han rat and the RUCA-II cell line derived from an endometrial adenocarcinoma of an inbred BDII/Han rat. The RUCA-I cell line, if transplanted to the neck of female DA/Han rats, gives rise to endometrial adenocarcinomas at the ectopic site. The morphology of these ectopically grown tumors is predominantly of the moderately differentiated sub-class. In contrast, ectopic tumor growth of the RUCA-II cell line can be observed only if cells are transplanted to athymic nude mice. Biochemically, both cell lines are characterized by the stable expression of estrogen receptors. However, no statistically significant mitotic response of RUCA-I and RUCA-II cells to estradiol was measurable, and no induction of expression of the progesterone receptor by estradiol was detectable, although estradiol transformed the estrogen receptor into its stable DNA-binding state. In contrast, the rate of proliferation of RUCA-I but not of RUCA-II cells was reduced in the presence of 10(-6) M tamoxifen. From these results we conclude that (i) both cell lines, RUCA-I and RUCA-II, represent a new and promising endometrial tumor model; (ii) the mechanism of the hormone-dependent growth regulation of RUCA-I and RUCA-II cells is obviously impaired; (iii) the RUCA-I cell line appears to be a suitable model system for the study of molecular aspects of estrogen- and tamoxifen-dependent gene expression.

Pharmacokinetics and protein binding of gestodene under treatment with a low-dose combination oral contraceptive for three months.

The serum concentrations of gestodene (CAS 60282-87-3) as well as the binding of this progestin to serum proteins were studied in 40 women who took a low-dose oral contraceptive (Femovan, Femodene) containing 30 micrograms ethinyl estradiol (CAS 57-63-6) and 75 micrograms gestodene for 3 months. On days 1, 10, and 21 of the first and the third treatment cycle, respectively, 7 blood samples were drawn before and up to 4 h after pill intake; additional samples were taken prior to morning ingestion of pill on days 2, 5, 11, 15 and 22 of these cycles. Gestodene levels were measured by means of a specific radioimmunoassay and were evaluated for Cmax, tmax, and AUC up to 4 and 24 h. Independent of the test day and the treatment cycle studied, mean maximum gestodene serum levels were found about 0.8 to 0.9 h after pill intake. During the first treatment cycle, mean values of Cmax, AUC(0-4h), and AUC(0-24h) amounted to 4.3 ng.ml-1, 9.3 ng.ml-1.h, and 27.3 ng.ml-1.h on test day 1; these values increased by 250-400% and by 300-500%, respectively, when days 10 and 21 were compared to day 1. On day 1 of the third treatment cycle, these pharmacokinetic parameters were higher by almost a factor of two as compared to the corresponding data obtained on the beginning of the first cycle whereas the increase of these values between day 1 and the subsequent test days (200-300%) was slightly lower in cycle 3 as compared to cycle 1.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: In Germany, health workers drew serum samples from 40 healthy young women who used a low dose oral contraceptive (OC) (Femovan, Femodene) with 30 mcg ethinyl estradiol and 75 mcg gestodene for 3 treatment cycles to measure gestodene levels, the free fraction of gestodene, and its distribution over the binding proteins in serum pools from all women. Mean maximum gestodene levels occurred .8 to .9 hours after taking the pill. The mean maximum gestodene level on test day 1 was 4.3 ng ml-1 during the first treatment cycle. This figure increased significantly to 7.4 ng ml-1 during the third cycle. Both day 1 levels were significantly lower than the levels obtained on day 10 (10.4 and 13.1 ng ml-1, respectively) and day 21 (12.1 and 13.4 ng ml-1, respectively). The area under the curve zero to 4 hours after pill intake was 9.3 ng ml-1. These figures rose 250-400% and 300-500%, respectively, on days 10 and 21. During the third treatment cycle, figures for days 10 and 21 were somewhat lower than those during the first cycle. 78% of total gestodene was bound to sex hormone binding globulin (SHBG) and 21% was bound to albumin on day 1 of the first treatment cycle. Just 1% of gestodene continued to be free. ON day 21, the fraction of gestodene bound to SHBG rose to 87%, that bound to albumin was 13%, and 0.5% remained free. Gestodene was not redistributed over its binding proteins during the third treatment cycle. These results indicated that pharmacokinetic accumulation and the increase in serum SHBG concentration and binding capacity for gestodene allows researchers to understand changes in gestodene levels during longterm treatment with a low dose OC containing gestodene.

Down-regulation of tenascin expression by antiprogestins during terminal differentiation of rat mammary tumors.

Studies on tenascin expression in hormonally dependent growing tissues of breast and endometrium suggested that its expression parallels the progression of normal or malignant proliferative alteration of the tissue. With the study presented here we addressed the question of whether antiprogestin-induced terminal differentiation down-regulates tenascin expression. By comparative immunolocalization of tenascin in sections of untreated 7,12-dimethylbenz[alpha]anthracene-induced tumors, tumors grown in ovariectomized animals, tamoxifen-treated tumors, and antiprogestin-treated tumors, we obtained the following results. (a) The entire extracellular space of the stromal mesenchyme was filled by tenascin immunoreactivity in cases of untreated control tumors. (b) Both ovariectomy and antiestrogen treatment with tamoxifen did not affect the overall staining pattern and resulted in a slight increase of the arbitrarily judged staining intensity. (c) Within antiprogestin-treated tumors tenascin-like immunoreactivity predominantly was restricted to fiber-like, collagenous connective tissue structures, which appeared in the stromal compartment as a result of the antiprogestin treatment. In large areas of the tumor composed of apparently secretory active tumor cells we failed to immunolocalize tenascin. Our results provide further evidence that expression of tenascin reflects both benign and malignant proliferative alterations of the tissue, whereas its down-regulation is correlated to differentiation of the tissue. Additionally, evidence is provided that the mechanism of tumor growth inhibition by antiprogestins indeed is induction of terminal differentiation of tumor cells.

[In vivo and in vitro effects of GnRH analogs on ovarian Leydig cell tumor]. / In-vivo- und In-vitro-Effekte von GnRH-Analoga auf einen ovariellen Leydigzelltumor.

A 65-year old patient, suspected to be suffering from an androgen producing ovarian tumour, was treated preoperatively with the GnRH agonist triptorelin (500 micrograms/day s.c.) for 7 days. After an initial rise, gonadotrophin levels were suppressed under this treatment. The elevated serum testosterone concentrations were reduced by approx. 50% by the triptorelin injections. After the extirpation of the tumour (histologically a Leydig cell tumour of the ovary without signs of malignancy), primary cell cultures which secreted testosterone and androstenedione were prepared. Coincubation of the tumour cells with the GnRH agonist triptorelin had no effect on their androgen secretion. Treatment of the tumour cells with high concentrations (10(-5) M) of a GnRH antagonist, however, resulted in a 100% increase of their testosterone and androstenedione secretion. GnRH-binding sites of low affinity (Ka = 0.54 x 10(5) M-1) and high capacity (B max = 1364 x 10(-12) M/mg membrane protein) were identified in the tumour. These findings suggest that GnRH analogues might modify androgen secretion of sex-cord stromal tumours of the ovary via the suppression of endogenous gonadotrophin secretion and possibly also via direct effects on the tumour cells.